A unique Toxoplasma gondii haplotype accompanied the global expansion of cats

Toxoplasma gondii is a cyst-forming apicomplexan parasite of virtually all warm-blooded species, with all true cats (Felidae) as definitive hosts. It is the etiologic agent of toxoplasmosis, a disease causing substantial public health burden worldwide. Few intercontinental clonal lineages represent the large majority of isolates worldwide. Little is known about the evolutionary forces driving the success of these lineages, the timing and the mechanisms of their global dispersal. In this study, we analyse a set of 156 genomes and we provide estimates of T. gondii mutation rate and generation time. We elucidate how the evolution of T. gondii populations is intimately linked to the major events that have punctuated the recent history of cats. We show that a unique haplotype, whose length represents only 0.16% of the whole T. gondii genome, is common to all intercontinental lineages and hybrid populations derived from these lineages. This haplotype has accompanied wildcats (Felis silvestris) during their emergence from the wild to domestic settlements, their dispersal in the Old World, and their expansion in the last five centuries to the Americas. The selection of this haplotype is most parsimoniously explained by its role in sexual reproduction of T. gondii in domestic cats.

In this complicated but fascinating manuscript, the authors report results from whole genome sequencing of Toxoplasma gondii isolates from around the world, and make inferences about the demographic history of this parasite based on the synthesis of genomic and historical evidence. In general, the analyses are competently conducted and the results are clearly explained, and will be of wide interest. The Discussion is speculative, but concordant with the genomic signals observed. The impact of the manuscript could be greater if accompanied by functional validation of the presumed selected T. gondii mutations conferring adaptation to domestic cats, but this work will presumably follow in a subsequent manuscript. This manuscript could be improved through clarification of a few issues: 1) It would be helpful to clarify which isolates are wild vs. domestic in all figures, most particularly figure 3. Further, it would be helpful if the authors addressed the asymmetry in sampling wild vs. domestic isolates from the New World vs. the Old World in their analyses; do any potential biases arise due to lack of sampling wild isolates in the Old World? Do prior studies of wild isolates in other felid species support or refute the conclusions drawn about adaptation to domestic cats? 2) Figure 4 is very difficult to understand, with many tiny elements. What is the main point of this figure, and could this be included in the title? It makes a complicated analysis more difficult to apprehend.
Small points: Toxoplasma is arguably the most successful parasite in the world, as a single species can infect all warmblooded animals and exists in a remarkable number of ecological niches. The vast majority of global clinical isolates (outside of S. America) come from one of three clonal lineages that are a familial clade, though the population is much more diverse in S. America. This assymetrical global population structure has often been argued to have been due to a founder's effect where a small number of strains "hitched" onto e.g. rats on colonial ships and then dispersed throughout the world. Given the large population diversity in S. America compared to the rest of the world, the most parsimonious model was often considered to be that Toxoplasma originated in e.g. Amazonian Brazil. The present study provides a massive new set of sequencing data to better define global population structure. These data, combined with the highest-quality estimate to-date of the mutation rate of parasites passed continuously through mice allowed the authors to argue that the parasite disseminated from Old World to New World rather than vice versa. The authors also argue that a small genomic region is correlated with "domesticated" parasite strains, which they suggest is therefore correlated with sexual recombination in domesticated (vs. wild) cats.
Overall, this is an intriguing paper, and the data will be incredibly useful to the community. Overall the analyses appear to have been carried out carefully. However, it appears that the authors set out to prove their model for maximum splash rather than merely analyze the data and see where it led them. While the authors argue that their model is most parsimonious, the argument does not convince this reviewer (which isn't to say the discussion isn't worthwhile or that I am unwilling to be convinced). While it is always important to a field to have long-held models disrupted, paradigm-shifting work must hold itself to the highest standards.
Major comments: -The vastly different recombination rates among the S American (frequent) vs. "dometicated" (incredibly rare; hence clonal populations) would be expected to complicate TMRCA estimates; one could not use the same mutational model for both populations. Also, the authors use Tang's equation to estimate TMRCA, but according to Tang's original publication one of the stated assumptions of Tang's analysis is that there is no recombination (fine in the clonal lineages, not so fine in the S. American population). This appears to call into question the accuracy of their estimates, upon which the majority of their arguments are based.
-The argument considered most parsimonious by the authors is that the clonal expansion of Toxoplasma is correlated with domestication of cats, i.e. genetic differences between dometicated vs. wild cats. But Ottoni et al (ref 40 in citation) show that very few genetic changes are correlated with cat domestication, and those that do vary are mostly in coat coloring. How could this have altered sexual recombination frequency? Also, Felis silvestris lybica and related species are still prevalent throughout the Mediterranean and Northern Africa, and North America is famous for large stretches of undeveloped land with many prevalent species of wild cats (for which e.g. the Canadian "cougar" strain is named from a scat-infected well), yet N America does not share the genetic diversity found in S. America -so does the argument hold that the major difference between Old World and New World Toxoplasma isolates being the loss of wild cat species? -More on the last point -since divergent S. American strains have been demonstrated to be able to superinfect and recombine experimentally in domestic cats, isn't that inconsistent with the authors' model? -Alternative hypothesis ignored by authors: Toxoplasma is unusual in that it does not require transmission through its definitive host to infect a new organism. Thus organisms that will eat meat, e.g. humans, hawks, rats (to some extent), can all get infected from eating an infected animal. Perhaps that plays a role in the "domestication" population.
Minor comments: -Labels in most figures are quite small and not legible on a printed page Reviewer #3 (Remarks to the Author): A unique Toxoplasma gondii haplotype accompanied the global expansion of cats. Galal et al.
This paper reports 156 Toxoplasma gondii genomes, of which 105 have not previously been analyzed at WGS resolution. The authors claim that they have identified a region on Chromosome Ia, which they refer to as a unique haplotype, that is common to clonal strains of Toxoplasma that infect and expand in domestic cats. This finding has been extensively published on previously (please see Khan, Genome Res, 2006;Khan, PNAS, 2007;Khan, mBio, 2011), just not at WGS resolution. The authors argue that by combining environmental and functional data, this region (which comprises 0.16% of the T. gondii genome) has been selected because it promotes sexual reproduction in domestic cats. But there is no experimental validation to back up this claim. They produced a direct estimate for the Toxoplasma gondii mutation rate. Based on SNP variation identified within 50 Type II isolates, they suggest that the clonal Type II lineage emerged 13K to 50K years ago, just prior to the domestication of cats. They argue that domestic T. gondii lineages carrying the same Chromosome Ia haplotype are more efficient at sexual expansion in domestic cats, but again, they do not provide the experimental data to prove this fact. Rather, they reference one paper that assayed a single wild strain of T. gondii that has a defect in self-mating as their evidence. Herein lies a fundamental problem with the author's interpretation, since the vast majority of wild strains are readily infectious in mice, produce transmissible cysts, and can be expanded as highly fecund infections in domestic cats (see Khan, PNAS, 2007; in addition to the vast collection of wild strains generated at the USDA by Dr. JP Dubey, who recovered a majority of these wild strains by assaying them through domestic cats). What is novel, is the use of Population Branch Statistics to further resolve the previously identified Chromosome Ia region to a limited set of polymorphic genes that should certainly be tested for their ability to promote sexual reproduction in domestic cats in an allele-specific manner to substantiate the author's claims. Without this, the paper fails to provide sufficient evidence to support this region as one potential explanation for the global expansion of specific T. gondii clonotypes by domestic cats.
Major Points: 1. The title is both misleading and insufficiently supported. The unique haplotype refers to a region on Chromosome Ia that is common to clonal lineages I, II, III. But this region is divergent in clonal lineage HG12, why was this not discussed? HG12 isolates produce highly fecund infections in domestic cats and are considered the 4th clonal lineage of North America. One study previously showed that Type II and HG12 cause essentially the same frequency of T. gondii infections in feral domestic cats on the West Coast of the USA (Van Wormer, 2014, PLoS NTD). These data certainly challenge their thesis that the region on Chromosome Ia common to Types I, II, III is responsible for the selective expansion of clonotypes through domestic cats. Further, the molecular clock has not been rigorously calculated, and is rife with assumption biases (see below), so it is impossible to accurately infer the timing of the molecular origin of the domestic Type II clonal lineage. Suggesting that it pre-dates the domestication and global expansion of cats is not based in fact, and borders on wishful thinking.
2. The authors identified 1,262,582 variant positions that were shared across the 156 genomes sequenced. They then performed what they called a "clone-censoring" step to remove 85 isolates (from  what I gather, it was 2 Type I, 49 Type II, 17 Type III, 14 Africa 1, 3 Africa 3), which reduced the number of variant positions to only 588,777 SNPs (or by 53%). The argument made was that diversity within a lineage is minimal, so this diversity would not influence the ancestry plots that showed extant admixture only among the non-clonal domestic strains from the Old and New World (Supplementary Figure 1). However, by removing the heterogeneity present among the clonal lines, the analysis presented a strongly biased and potentially artefactual interpretation of the data. Figure 2 clearly suggests that significant diversity exists within clonal strains Type II, III and Africa 1, although no scale was provided in the figure, so it is impossible to gauge the true number of SNP differences among strains within each lineage. What happened to ~674,000 SNPs? Supplementary Figure 6 establishes that admixture among domestic Type II, Type III and Africa 4 clonal strains has occurred, which would surely affect the TMRCA calculations. If all "clone-censored" strains are included, how does this affect the "timing" of the molecular origin of the clonotypes with respect to the domestication of cats? 3. Figure 3 needs to be radically overhauled. The ancestry plots suggest that Type I, II, III and Africa 4 do not exist as admixture clones, when in fact multiple studies (Boyle PNAS, 2006;Lorenzi, NatComm, 2016;Zhang, 2017, Mol Biol Evol) clearly establish these lineages as admixtures. Hence, the single color hue across each chromosome by clonotype belies truth. For example , Type II shares chromosomes Ia and IV  with Type I strains, and Type II shares chromosomes Ia, XI, and parts of Ib, III, VI, VIIb, VIII, IX and XII with Type III. Figure 3 needs to be re-imagined, it fails to reflect truth, each clonal strain is an admixture of different colors. The cut-offs also do not coincide with the analyses performed in Figure 4 and Supplemental Table 4. For example, the HG12 strains WdUS01 and WdUS04 share the same color hue (green; Type II) on chromosome III with two recombinant strains (WdUS02, WdUS03), but the analysis presented in Supplemental Table 4 for these 4 isolates suggest that the two HG12 strains are at least 47K-177K years distant from the two recombinant strains that share the same color at this chromosome. This one example calls into question the entire dataset.
4. Molecular Clock calculation. The authors do not possess reliable data on the passage history for the strains they used to estimate natural variation. Lab strains separated by 30 years, with no reliable estimate for the true number of passages they have undergone, nor what manipulations they have been exposed to, is not reliable. If the authors want to include such data, they need to carefully record the number of passages between each line and sequence multiple clones from independent cultures across defined time points to truly achieve a reliable result, as has been done previously for Plasmodium. In their own words, they state (line 267; TMRCA estimates are very sensitive to sampling and obtaining accurate estimates depends on robust sampling of source populations). Too many unreliable estimates are extant in the literature, and the authors have the methodology in place to do this precisely. Furthermore, how does their result compare to other TMRCA calculations performed for T. gondii based on examining drift in introns across a large number of T. gondii strains? 5. The data estimates presented in Figure 4 suggest that the time between the two most divergent genomes of Type II is significantly greater than for the other clonotypes, which they argue are more recently derived. However, this result more likely reflects their bias in sampling. They had 48 Type II strains to draw from, whereas they only had 3 Type I strains (Supplemental Table 8). Importantly, there existed about 500 SNPs between the 3 Type I strains. Assuming the accumulation of SNPs is similar among the different clonotypes (across time), 3 goes into 48 sixteen times (or 500x16=8000 SNPs) which is nearly equivalent to the number of SNPs that separate the two most divergent Type II lineages. Which may suggest that Type I is as OLD as Type II and that the Figure 4 analysis is potentially inaccurate and prone to an oversampling bias.
6. The penetrance of the Chromosome Ia haplotype among successful strains of T. gondii that expanded to represent a majority of infections in North America and Europe is certainly interesting (and has been identified previously). While it is possible that the PBS analysis performed using the strains in this study support a region on Chromosome !a as potentially relevant for the success of these strains, the authors failed to prove that a gene in this region is responsible for the expansion of these strains among domestic cats, as they strongly suggest. Importantly, this analysis fails to include a large number of HG12 isolates, that are likewise highly successful clones, that produce highly fecund infections in domestic cats, but do not share the same chromosome Ia haplotype. It is therefore incumbent on the authors to provide formal proof, that a gene in this region is both necessary and sufficient to promote sexual reproduction in domestic cats to rationalize the success of these clonotypes, as they report.
Minor Points: 1. Which is it? For Type II, Fig 2 lists 47 isolates, Supplemental Table 1 lists 50 isolates, Supplemental  Table 8 lists 48 isolates. Please be consistent. If strains are dropped from particular analyses, a rational should be provided.
2. Line 278 -"…TMRCA estimates indicate that massive introgressions of types I, II, II and Africa 1 into New World populations have occurred…" I think the authors are suggesting here that Old World lineages have recombined multiple times with New World strains to produce admixtures lines that exist as chimeras -is that correct?
3. The authors renamed the vast majority of strains to conform with their "domestic" vs. "wild" designation and geographic origin of the isolate. This was quite a distraction, to sort out which designation was GT1, Me49, VEG, etc. The organization of the Supplemental Tables was difficult to infer. Is it possible to add the common name to Supplemental Table 2, and also re-sort Supplemental Table 2 to be by multilocus Type, then by country? Or if the preference is to sort by country, then please sort by multilocus type.
4. In the Supplementary File, line 383, the authors state Type III genomes (n=19), but in all other places within the manuscript, they state that only 18 Type III genomes were analyzed -which is it? 5. The authors propose a model in which rodents in the New World are multiply co-infected with different strains of T. gondii to support the increased diversity detected in isolates from the New World. Do the authors have evidence to support such a claim, or have studies been done that they can reference that indicate such a high frequency of mixed infections to support their proposed model?

Reviewer #1 (Remarks to the Author):
1 2 In this complicated but fascinating manuscript, the authors report results from whole genome 3 sequencing of Toxoplasma gondii isolates from around the world, and make inferences about the 4 demographic history of this parasite based on the synthesis of genomic and historical evidence. In 5 general, the analyses are competently conducted and the results are clearly explained, and will be of 6 wide interest. The Discussion is speculative, but concordant with the genomic signals observed. The 7 impact of the manuscript could be greater if accompanied by functional validation of the presumed 8 selected T. gondii mutations conferring adaptation to domestic cats, but this work will presumably 9 follow in a subsequent manuscript. This manuscript could be improved through clarification of a few 10 issues: 11 AUTHORS' RESPONSE -We are grateful to the reviewer for accepting to revise our work and we 12 thank him for his positive remarks. 13 1) It would be helpful to clarify which isolates are wild vs. domestic in all figures, most particularly 14 figure 3. 15 AUTHORS' RESPONSE -We modified Fig. 3  None of the two seronegative margay cats (Leopardus wiedii) challenged with the same strain 50 excreted oocysts. However, one seronegative cougar (Puma concolor) and one seronegative 51 Jaguarondi (Puma yagouaroundi) challenged with this strain excreted oocysts. Interestingly, two 52 Asian leopard cats (Prionailurus bengalensis) excreted oocysts after being challenged with LRH strain, 53 but not after a previous challenge with M-7741 strain. LRH strain was previously isolated from the 54 faeces of a Panamanian ocelot (Felis pardalis), and is therefore probably a true wild strain (Jewell et 55 al., 1972) but this is not sure of course. Overall, these results suggest that domestic strains are poorly 56 transmitted by wilds felids but it is for now impossible to demonstrate clear species-specific patterns 57 based on these small samples. In organisms having frequent recombinations (humans for example), a genomic sequence can be a 64 succession of very small portions of sequences having different ancestries due to the accumulation of 65 ancestry mixing by crossovers through generations. This obviously complicates the calculations of 66 TMRCA, and therefore many different sophisticated models have been developed to resolve this 67 issue. This is not the case in our study as recombinations in the here considered T. gondii populations 68 are infrequent. Many strains have inherited "intact" chromosomes of single ancestry (Fig.3) showing 69 no evidence of crossovers on them. These chromosomes, although inherited following a process of 70 sexual reproduction, are clones of one of their respective parental strains. 71 Our objective was to demonstrate that (1) intercontinental lineages recently spread from the Old 72 World to the New World and that (2) domestic New World strains are the progeny of these Old 73 World lineages? To do so, we first identified all chromosomes having a single ancestry among 74 putative hybrids from the plots on figure 3. We used these "intact" chromosomes of single ancestry 75 and estimated their divergence (TMRCA) from Old World lineages. For example, if a domestic 76 Brazilian strain had a full ancestry of type III at chromosome 2 (blue in Fig. 3), we compared the 77 sequence of this Brazilian chromosome to all chromosome 2 sequences of all types III strains from 78 the Old World. Finding a TMRCA of ~500 years would support the two hypotheses mentioned in the 79 beginning of this paragraph. In the figure, the different colours represent the different ancestries and 80 the lengths of ellipses correspond to the interval of TMRCA (e.g. 600-300 years). Toxoplasma is arguably the most successful parasite in the world, as a single species can infect all 97 warm-blooded animals and exists in a remarkable number of ecological niches. The vast majority of 98 global clinical isolates (outside of S. America) come from one of three clonal lineages that are a 99 familial clade, though the population is much more diverse in S. America. This assymetrical global 100 population structure has often been argued to have been due to a founder's effect where a small 101 number of strains "hitched" onto e.g. rats on colonial ships and then dispersed throughout the world. 102 Given the large population diversity in S. America compared to the rest of the world, the most 103 parsimonious model was often considered to be that Toxoplasma originated in e.g. Amazonian Brazil. 104 The present study provides a massive new set of sequencing data to better define global population 105 structure. These data, combined with the highest-quality estimate to-date of the mutation rate of 106 parasites passed continuously through mice allowed the authors to argue that the parasite 107 disseminated from Old World to New World rather than vice versa. The authors also argue that a 108 small genomic region is correlated with "domesticated" parasite strains, which they suggest is 109 therefore correlated with sexual recombination in domesticated (vs. wild) cats. 110 Overall, this is an intriguing paper, and the data will be incredibly useful to the community. Overall 111 the analyses appear to have been carried out carefully. However, it appears that the authors set out 112 to prove their model for maximum splash rather than merely analyze the data and see where it led 113 them. While the authors argue that their model is most parsimonious, the argument does not 114 convince this reviewer (which isn't to say the discussion isn't worthwhile or that I am unwilling to be 115 convinced). While it is always important to a field to have long-held models disrupted, paradigm-116 shifting work must hold itself to the highest standards. 117 AUTHORS' RESPONSE -We thank the reviewer for the positive remarks about the interest of our 118 manuscript and we are grateful to him for his sincere will to help in improving the consistency and 119 the robustness of the conclusions drawn from our results. 120 We regret this impression of "maximum splash" as it was not at all our intention. In the revised 121 version of the manuscript, we fully restructured the discussion. We anchor it on the major findings of 122 our study in a more systematic way before developing our interpretation. 123 Before answering to each of the reviewer's specific comments, we would like to comment some of 124 the assertions developed in his opening paragraph. 125 The reviewer's argues that the most parsimonious model was often considered to be that 126 Toxoplasma gondii originated in South America and that our study is disrupting a long-held model. 127 First, we would like to point that the objective of our study was to focus on recent history of T. gondii 128 in relation to domestication (see l.108-109). It was not designed to answer to the question of the 129 origin of T. gondii. We do not argue that the parasite disseminated from Old World to New World, 130 but rather that the domestic haplotype of T. gondii followed this route. In addition, our results 131 provide some insights about the genetic proximity between type II, Chinese 1 and haplogroup 12. By 132 crossing our TMRCA estimates (late Pleistocene) to historical data on animal migration at this time, 133 we hypothesized that T. gondii migrations took place from Asia to North America at this time and not 134 the opposite. This hypothesis is specific to T. gondii strains of clade D (type II, Chinese 1 and 135 haplogroup 12). However, we do not pretend at all to provide evidence for an Old World origin of T. whereas other studies proposed an origin in North America Khan et al. (2007) PNAS ; Minot et al. 140 (2012) PNAS. T. gondii emerged in the wild several millions years ago. Therefore the history of its 141 origin is a strictly wild history and needs wild isolates to be understood. With the benefit of hindsight, 142 we think today that the absence of wild strains from the Old World in all above mentioned studies 143 (only wild strains from North and South America were available) does not enable to answer to the 144 question of the origin at this stage. That is why we are only addressing the question of recent history 145 in the present study. 146 Major comments: 147 -The vastly different recombination rates among the S American (frequent) vs. "domesticated" 148 (incredibly rare; hence clonal populations) would be expected to complicate TMRCA estimates; one 149 could not use the same mutational model for both populations. 150 AUTHORS' RESPONSE -Genetic evidence (from this study and from previous ones) supports various 151 degrees of clonality among different populations (according to geography and to ecotype). However, 152 we do not have precise knowledge of the recombination rate of Toxoplasma gondii in the literature. 153 With all the respect due to the reviewer, we do not agree with its distinction between "the S 154 American (frequent) vs. "domesticated" (incredibly rare; hence clonal populations)" for two reasons: 155 (1) Domestic clonal populations are common in South America (e.g. Caribbean 1,2,3) so opposing 156 South American strains to domesticated strains or to clonal populations is not relevant in our 157 opinion.
(2) Our local ancestry analyses (Fig.3) clearly show that recombinations are rare in domestic 158 South American populations. Indeed, we do not observe a fine mosaic of different ancestries 159 alternating across these genomes but we rather observe large blocks (often reach the full length of 160 chromosomes) of single ancestry, a pattern indicative of rare crossovers. By considering the genomic 161 pattern of recombination obtained from a single experimental recombination between two T. gondii 162 strains (Khan et al., 2014b BMC Genomics;FIG.3), it appears that the ancestry pattern we have in our 163 study ( Fig. 3) is consistent with the idea that few rounds of recombination were sufficient to give rise 164 to South American domestic populations. However, we agree that the recombination rate of these 165 populations appears to be greater than that of domestic populations in North America, and also that 166 of Old World populations, for which recombinations are "incredibly rare" events. 167 The question of the methodology used in TMRCA estimation is of course crucial. 168 We all know that crossovers occurring between two sequences during recombination result in a new 169 sequence which is a mosaic of the two parental sequences, and these parental sequences often have 170 very different histories. Now if we calculate the TMRCA separating this new sequence from other 171 sequences, this could not be straightforward since the TMRCA will vary according to the portion of 172 the new sequence that is considered (portions inherited from parent 1 or parent 2). In organisms 173 having frequent recombinations (humans for example), a genomic sequence can be a succession of 174 very small portions of sequences having different ancestries due to the accumulation of ancestry 175 mixing by crossovers through generations (some examples can be found in these studies Henn et al., 176 2012;Fitak et al., 2018;Kim et al., 2020). This obviously complicates the calculations of TMRCA, and 177 therefore many different sophisticated models have been developed to resolve this issue. 178 This is not the case in our study as recombinations in the here considered T. gondii populations are 179 infrequent. Indeed, many strains have inherited "intact" chromosomes of single ancestry (Fig.3)  180 showing no evidence of crossovers on them. These chromosomes, although inherited following a 181 process of sexual reproduction, are clones of one of their respective parental strains. We only used 182 these "intact" chromosomes in our TMRCA calculations. 183 Also, the authors use Tang's equation to estimate TMRCA, but according to Tang Also, Felis silvestris lybica and related species are still prevalent throughout the Mediterranean and 220 Northern Africa, and North America is famous for large stretches of undeveloped land with many 221 prevalent species of wild cats (for which e.g. the Canadian "cougar" strain is named from a scat-222 infected well), yet N America does not share the genetic diversity found in S. America -so does the 223 argument hold that the major difference between Old World and New World Toxoplasma isolates 224 being the loss of wild cat species? 225 AUTHORS' RESPONSE -The question of the reviewer here is of course a crucial element for the 226 consistency of the paradigm we develop in our study and we are therefore grateful to him for asking 227 this question. In the recent review of T. gondii genotypes in North America (Jiang et al., 2018 FIG .1), 228 we can see that many genotypes are still persisting in the wild in this continent. Of course the ~ 70 229 million domestic cats found in North America have huge environmental impact, with oocysts shed by 230 these domestic hosts reaching wildlife (even marine wildlife) by spreading over long distances via 231 waterways (Dabritz and Conrad, 2010;VanWormer et al., 2013a). This explains the high frequency of 232 domestic genotypes (type II, type III) among wildlife from this continent. 233 Also, important heterogeneity exists within South America when we compare wild isolates from 234 different countries of the continent. It is clear that the most diversified population of T. gondii 235 described to date is occurring in the Amazonian forest of French Guiana, a relatively well-preserved 236 environment with 8 species of wild felids cohabiting in this environment, which is a quite unique 237 situation (Mercier et al., 2011). The wild environment is far less preserved in many regions of South 238 America. In Brazil for example, strains isolated from wild animals were often also found in domestic 239 animals. One could argue that these environmental interpenetrations of strains could blur the signal 240 of the distinction between domestic and wild strains as we are presenting it in our study. What we 241 clearly see is that finding a strain in the domestic environment not harbouring the cat's adaptation 242 haplotype is exceptional and such strains fail to occupy domestic niches even in situations where 243 domestic and wild environment are in close contact (e.g. French Guiana). 244 We clarify these points in the revised manuscript. 245 We hope the previous answer provide enough clarification regarding the specific example of Felis and "divergence" is of course a relative concept. According to our results and to previous results (Su 255 et al., 2012;Lorenzi et al., 2016), it is clear for example that wild Amazonian strains are highly 256 divergent from the major domestic types (types I,II,III and Africa 1). Our results also show that wild 257 type 12 strains (RFLP lineage #5) from North America are "moderately" divergent from type II strains. 258 Importantly, domestic South American strains and wild South American strains should not be 259 considered one and the same, as the latter are a mixture of wild South American strains and major 260 domestic lineages. Domestic South American strains have inherited large portions of their genome 261 from types I,II,III and Africa 1 and this happened very recently according to our dating estimates. 262 Cross-immunity between these two groups is therefore likely to occur, but this will likely be 263 determined by the inheritance (or not) of alleles of genes involved in immunological recognition. 264 Anyway, it is important to recall that superinfected hosts were rarely found in real life. This rarity fits 265 well with our model, which supports rare recombinations (see Fig.3), knowing that rare 266 recombinations are probably the result of rare superinfections. 267 Experimental results suggest that wild strains are less efficiently excreted in the form of oocysts by 268 domestic cats compared to domestic strains (Khan et al., 2014a). Therefore, sexual reproduction 269 involving a wild strain in a domestic cat is probably a rare event in natural conditions. Again, this 270 rarity fits well with our model, which supports rare recombinations (more frequent than in other 271 parts of the World though). 272 Alternatively, if we assume that most domestic South American strains are effective at frequently 273 superinfecting hosts, we are breaking a major barrier for sex in the T. gondii cycle, and there will 274 likely be much less clonality in South American domestic populations, which is not the case (Pena et 275 al., 2008;Mercier et al., 2011). 276 277 -Alternative hypothesis ignored by authors: Toxoplasma is unusual in that it does not require 278 transmission through its definitive host to infect a new organism. Thus organisms that will eat meat, 279 e.g. humans, hawks, rats (to some extent), can all get infected from eating an infected animal. 280 Perhaps that plays a role in the "domestication" population. at WGS resolution. 299 The authors claim that they have identified a region on Chromosome Ia, which they refer to as a 300 unique haplotype, that is common to clonal strains of Toxoplasma that infect and expand in domestic 301 cats. This finding has been extensively published on previously (please see Khan, Genome Res, 2006;302 Khan, PNAS, 2007;Khan, mBio, 2011), just not at WGS resolution. 303 AUTHORS' RESPONSE -We are grateful to the reviewer for accepting to revise our work. 304 The reviewer questions the novelty of our results compared to what has been published previously. 305 We are mentioning the contribution of Khan et al. in our manuscript l.359-360: "Note that a number 306 of strains shared the same haplotype for the whole length of chromosome 1a, a pattern previously 307 noticed in past studies" and we are citing the main paper on this topic (Khan et al., 2007 PNAS). 308 Khan et al. noticed that many strains shared what they named "a monomorphic version of 309 chromosome 1a" (same allele for the whole length of chromosome 1a using several short markers). 310 They proposed a number of hypotheses (among them the hypothesis of adaptation to domestic 311 environment) to explain this pattern. However, they could not show that this "monomorphic version 312 of chromosome 1a" is a marker of adaptation to the domestic environment for the very simple 313 reason that although many domestic strains harboured this monomorphic version of the whole 314 chromosome 1a, many also did not (Khan et al., 2007 PNAS;Khan et al., 2011 mbio). our manuscript. Our study shows that this pattern can be explained by the much stronger linkage 331 disequilibrium (often reaching the whole length of chromosome 1a) observed in domestic strains 332 relative to wild ones around the ~100 kb outlier region, which probably persists due to the combined 333 effects of selection and rarity of sexual recombinations in domestic T. gondii populations. Since the 334 conserved domestic haplotype is only a portion of chromosome 1a, we show that the appellation 335 "monomorphic version of chromosome 1a" used in several publications is inaccurate and misleading. 336 Having regard to these elements, we can assert that we have shown for the first time that we have 337 identified a "unique global haplotype common to almost all domestic strains worldwide and that is 338 under strong positive selection in the domestic environment". We believe that this is a remarkable 339 finding. Moreover, by accurately defining the genomic boundaries of the global domestic haplotype, 340 we are able for the first time to propose a limited number of candidate genes (based on several 341 criteria) that could be involved in adaptation of T. gondii to the domestic environment. We are filling these gaps in this study by analysing the largest dataset of T. gondii genomes produced 365 to date (n=156) and by providing for the first time a direct estimate of T. gondii mutation rate and an 366 estimate of its generation time. We therefore reconstruct the recent history of global dissemination 367 of the domestic haplotype of T. gondii based on the basis of much more solid data (whole genomes) 368 and sampling. 369 The authors argue that by combining environmental and functional data, this region (which 370 comprises 0.16% of the T. gondii genome) has been selected because it promotes sexual 371 reproduction in domestic cats. But there is no experimental validation to back up this claim. 372 AUTHORS' RESPONSE -We partially agree with the reviewer on this point. 373 Full evidence to prove the selection of a trait should be supported by genomic patterns, geographic 374 distributions, and functional characterization. We will discuss each of these three aspects to show 375 how our results and previous results support our point. 376 Genomic evidence: Genomic evidence is supported by the results provided in Figure 5 (Population 377 Branch Statistics, high degree of conservation of the domestic haplotype, extended linkage 378 disequilibrium around domestic SNPs). 379 Geographic evidence: Geographic evidence is supported by the tight association between the 380 domestic haplotype of T. gondii and the environment of domestic cats (and its evolution in time, 381 mainly with the coincidence in the estimates between the introduction of domestic cats in the New 382 World and the emergence of domestic New World strains harbouring the domestic haplotype of T. 383 gondii). 384 In the domestic environment, other hosts can get infected but few play a role in transmission, as T. 385 gondii cycle is essentially based on transmission between cats and their prey (mainly rodents, then 386 birds). A host must be efficiently transmitting a parasite to be able to exert a selective pressure on 387 this parasite. A paper (Müller, Urs B., and Jonathan C. Howard. "The impact of Toxoplasma gondii on 388 the mammalian genome." Current opinion in microbiology 32 (2016)) discusses very nicely the idea 389 of difference in host importance and explains the notion of "evolutionarily significant host". In this 390 regard, the domestic cat have a tremendous advantage over any other host given its ability to 391 excrete dozen of millions of highly resistant oocysts that survive during months in the environment. 392 House mice and rats are important hosts to consider in this frame as they are important prey of cats 393 and have also recently spread to many parts of the world (the New World and West Africa). It has 394 been suggested that house mice could play a role in selecting certain T. gondii strains at the expense 395 of others. However, selection exerted on T. gondii by mice was associated to the virulence of T. 396 gondii strains, which is not explained by the opposition between wild and domestic environments 397 since many mouse-virulent strains are found in both environments (Howe and Sibley, 1995;Pena et 398 al., 2008;Mercier et al., 2010Mercier et al., , 2011Shwab et al., 2018). Moreover, house mice and rats are not 399 found in certain areas where the domestic haplotype of T. gondii is well established so the 400 geographical association does not stand for these species (Dalecky et al., 2015;Hima et al., 2019 the boundaries of the genomic region under selection, we are able for the first time to propose a 417 limited number of candidate genes. In addition, two recent studies (Ramakrishnan et al., 2019;418 Farhat et al., 2020) have provided accurate data regarding the stage of expression of these genes. 419 This important contribution enabled to determine which genes on the ~100 kb haplotype are 420 expressed during sexual reproduction of T. gondii in cats. By crossing these findings to our results, we 421 were able to shed light on the few most promising genes within this haplotype to explain this 422 function. 423 We are clarifying these elements in the revised manuscript. 424 Finally, we think it is important to recall the fact that this is a study of population genomics and not a 425 mechanistic study, although it provides valuable data for future purely mechanistic studies. 426 They produced a direct estimate for the Toxoplasma gondii mutation rate. Based on SNP variation 427 identified within 50 Type II isolates, they suggest that the clonal Type II lineage emerged 13K to 50K 428 years ago, just prior to the domestication of cats. They argue that domestic T. gondii lineages 429 carrying the same Chromosome Ia haplotype are more efficient at sexual expansion in domestic cats, 430 but again, they do not provide the experimental data to prove this fact. 431 AUTHORS' RESPONSE -In order to avoid lengthy repetitions, we invite the reviewer to refer to our 432 response to its second comment, in which we have already addressed precisely this concern. 433 Rather, they reference one paper that assayed a single wild strain of T. gondii that has a defect in 434 self-mating as their evidence. 435 AUTHORS' RESPONSE -With all the respect due to the reviewer, this is not the case. This study 436 performed experiments on domestic cats by testing four domestic strains versus three wild strains 437 (Khan et al., 2014a). 438 Herein lies a fundamental problem with the author's interpretation, since the vast majority of wild 439 strains are readily infectious in mice, produce transmissible cysts, and can be expanded as highly 440 fecund infections in domestic cats (see Khan, PNAS, 2007; in addition to the vast collection of wild 441 strains generated at the USDA by Dr. JP Dubey, who recovered a majority of these wild strains by 442 assaying them through domestic cats). 443

AUTHORS' RESPONSE -This is an important point. 444
In the beginning, we would like to recall that the hypothesis of adaptation of domestic T. gondii 445 strains to sexual transmission by domestic cats have been proposed for the first time by Dr. JP Dubey 446 himself (Khan et al., 2014a). 447 The reviewer argues that "the vast majority of wild strains are readily infectious in mice, produce 448 transmissible cysts". We totally agree with this statement but it is just not relevant in our case as we 449 are questioning in our study the different abilities of T. gondii strains to be transmitted by domestic 450 cats in the form of oocysts. 451 The reviewer argues that "the vast majority of wild strains […] can be expanded as highly fecund 452 infections in domestic cats" and is citing Khan, PNAS, 2007. But we did not find any element to 453 support this statement in the study he is mentioning which did not address this topic. 454 The reviewer evokes "a vast collection of wild strains" belonging to Dr. Dubey .We are not aware of 455 this vast collection but we know from the articles published by Dr. Dubey that wild strains may not 456 be transmitted by domestic cats and even more: domestic strains may not be transmitted by 457 domestic cats Khan et al., 2014a). So this question should be addressed as 458 a question of differences in transmission efficiency and this what the study by Khan et al., (2014a)  We are providing several references supporting this idea in the revised manuscript. 486 What is novel, is the use of Population Branch Statistics to further resolve the previously identified 487 Chromosome Ia region to a limited set of polymorphic genes that should certainly be tested for their 488 ability to promote sexual reproduction in domestic cats in an allele-specific manner to substantiate 489 the author's claims. Without this, the paper fails to provide sufficient evidence to support this region 490 as one potential explanation for the global expansion of specific T. gondii clonotypes by domestic 491 cats. 492 AUTHORS' RESPONSE -In order to avoid lengthy repetitions, we invite the reviewer to refer to our 493 response to its second comment, in which we have already addressed precisely this concern. 494 Major Points: 495 1. The title is both misleading and insufficiently supported. The unique haplotype refers to a region 496 on Chromosome Ia that is common to clonal lineages I, II, III. 497 AUTHORS' RESPONSE -By only considering the Figure 5d (now Figure 4d), we can clearly see that the 498 unique haplotype is common to all domestic strains (except one) so we cannot agree with this 499 assertion. Almost all domestic strains (grey labels) cluster together within the NJ tree not only types 500 I,II and III . 501 But this region is divergent in clonal lineage HG12, why was this not discussed? HG12 isolates 502 produce highly fecund infections in domestic cats and are considered the 4th clonal lineage of North 503 America. 504 AUTHORS' RESPONSE -We are grateful to the reviewer for bringing this point to our attention and 505 this is of course a very important question. 506 HG12 (as its name suggests) is not a clonal lineage but a haplogroup, composed of at least two major 507 clonal lineages (ToxoDB #4 and #5) and a number of minor lineages, all found in North America. So it 508 is a mistake to consider this haplogroup as a single homogenous entity (and we did this mistake). This 509 is important because the different lineages composing HG12 do not share the same allele for the 510 haplotype on chromosome 1a. 511 We re-examined the specific case of HG12 and what we found was very interesting and nicely 512 illustrates the association between the domestic T. gondii haplotype and the domestic environment. 513 The initial studies identified HG12 mainly among wild animals and therefore HG12 was first 514 considered as a wild haplogroup (Khan et al., 2011a). However, subsequent studies revealed that 515 HG12 was also common in the domestic environment. Jiang et al. (2018) published a very good 516 review of the genotypic diversity in North America which accurately reveals how the different 517 genotypes of T. gondii are distributed in the environment. The remarkable pattern we noticed about 518 HG12 in this review is that ToxoDB#4 is common in the domestic environment and rare in the wild, 519 whereas ToxoDB#5 shows the exactly opposite pattern. Our genomic analyses showed that 520 ToxoDB#4 (WdUSA02) harbours the domestic haplotype whereas ToxoDB#5 (WdUSA01 and 521 WdUSA04) harbours a wild haplotype. Although, we only have 3 strains representing these two 522 lineages, our results indicate that previous multilocus classifications (RFLP or MS) correlate strongly 523 (95%) with genomic classification. 524 Thus the pattern we observe for HG12 is one of the strongest supports to our hypothesis. This 525 example is very interesting in that it shows how two related lineages (#4 and #5) belonging to the 526 same haplogroup and found in the same region of the world segregate in space according to the type 527 of environment (domestic versus wild) and this segregation matches nicely with the different 528 haplotypes they harbour on chromosome 1a. 529 We have clarified this point in the new version of the manuscript. We renamed WdUSA02 (now 530 DcUSA11) to fit with this environmental evidence. 531 One study previously showed that Type II and HG12 cause essentially the same frequency of T. gondii 532 infections in feral domestic cats on the West Coast of the USA (Van Wormer, 2014, PLoS NTD). These 533 data certainly challenge their thesis that the region on Chromosome Ia common to Types I, II, III is 534 responsible for the selective expansion of clonotypes through domestic cats. 535 AUTHORS' RESPONSE -The authors of this study published a number of studies conducted in a very 536 interesting environment in which we observe an overlap (this is the term used by the authors) 537 between the home ranges of domestic cats and wild felids in this area. This environment is 538 contaminated with oocysts shed by both domestic cats and wild felids and therefore these two 539 groups of species can get infected with domestic and wild strains and this is what this study is 540 showing. However, the authors do not address the crucial question of whether or not domestic cats 541 infected in this area with wild strains excrete oocysts following their infection. The same authors are 542 asking this question in the discussion of their most recent paper on the subject (Shapiro et al., 2019 543 Proceedings of the Royal Society B), we quote "further studies on T. gondii oocyst genotypes shed by 544 domestic and wild felids would provide additional insight on sources of sea otter infection. While 545 Type X infections occur in both domestic and wild felids in watersheds bordering the sea otter range, 546 genotype data are needed for the oocysts shed by these felids. In experimental studies, the 547 prevalence of oocyst shedding varied with T. gondii strain. Greater levels of shedding were observed 548 in wild felids exposed to atypical 'wild' strains and in domestic cats exposed to archetypal 'domestic' 549 strains (e.g. Types I, II or III) [39,40], but only limited genotypes were tested. One of six domestic cats 550 experimentally infected with an atypical strain shed similar numbers of oocysts (2 × 108) as cats 551 infected with domestic strains [40]. To our knowledge, shedding of Type X oocysts by a domestic cat 552 has only been reported for one clinically ill animal [41]. Field studies are therefore needed to clarify 553 levels of shedding by domestic cats infected with Type X under natural conditions." Note: type X 554 refers to HG12. Therefore we can conclude that all elements brought by these studies support our 555 model. 556 Further, the molecular clock has not been rigorously calculated, and is rife with assumption biases 557 (see below), so it is impossible to accurately infer the timing of the molecular origin of the domestic 558 Type II clonal lineage. Suggesting that it pre-dates the domestication and global expansion of cats is 559 not based in fact, and borders on wishful thinking. 560 AUTHORS' RESPONSE -As the reviewer is developing his concerns about our estimates of TMRCA in 561 comment number 4, we will provide a detailed response on this point in our answer to comment 562 number 4. 563 2. The authors identified 1,262,582 variant positions that were shared across the 156 genomes 564 sequenced. They then performed what they called a "clone-censoring" step to remove 85 isolates 565 (from what I gather, it was 2 Type I, 49 Type II, 17 Type III, 14 Africa 1, 3 Africa 3), which reduced the 566 number of variant positions to only 588,777 SNPs (or by 53%). The argument made was that diversity 567 within a lineage is minimal, so this diversity would not influence the ancestry plots that showed 568 extant admixture only among the non-clonal domestic strains from the Old and New World 569 (Supplementary Figure 1). However, by removing the heterogeneity present among the clonal lines, 570 the analysis presented a strongly biased and potentially artefactual interpretation of the data. 571 AUTHORS' RESPONSE -Here we have followed basic recommendations to minimize bias related to 572 clonality for ADMIXTURE, a software developed for sexual organisms. If we include all strains (even 573 strains of the same clonal lineages), this will introduce huge bias in the analysis by deviating widely 574 from the assumption of sexual reproduction. The developers of this software also recommend 575 performing pruning of data for linkage disequilibrium (see: 576 https://vcru.wisc.edu/simonlab/bioinformatics/programs/admixture/admixture-manual.pdf). 577 According to the developers (see 2.4.), it is not uncommon that only few dozens of thousands SNPs 578 are retained after this step and this relatively small number of SNPs still provides enough resolution. 579 One should care on minimizing bias to fit as much as possible with the assumptions of the software's 580 model much more than the number of SNPs. 581 Figure 2 clearly suggests that significant diversity exists within clonal strains Type II, III and Africa 1, 582 although no scale was provided in the figure, so it is impossible to gauge the true number of SNP 583 differences among strains within each lineage. 584 What happened to ~674,000 SNPs? 587 AUTHORS' RESPONSE -As we pointed above, these SNPs were eliminated in certain analyses to 588 remove bias related to clonality when relevant. 589

AUTHORS' RESPONSE -This information is provided in Supplementary
Supplementary Figure 6 establishes that admixture among domestic Type II, Type III and Africa 4  590 clonal strains has occurred, which would surely affect the TMRCA calculations. 591 AUTHORS' RESPONSE -We can see on some positions with numerous SNPs on chromosome 10 of 592 type II strains, but this is not due to recombination as we observe no recombination breakpoints. 593 Chromosome 10 of type II strains: 594 595 For Africa 4 and type III, we invite the reviewer to reread the section "identifying clonal lineages" in 596 the supplementary information. As we have clarified in this section, "We therefore excluded the 597 divergent genomes from their respective poppr-defined lineages -which are likely the products of a 598 recombination with a strain of a distinct population-and generated new SNPs density plots .The 599 sharp variations in SNPs densities previously observed did not recur in the new plots (data not 600 shown), indicating that the excluded genomes had divergent ancestry in certain chromosomal 601 portions." 602 For type III strains we clearly noticed the recombination breakpoints before removing DcUSA04. 603 If all "clone-censored" strains are included, how does this affect the "timing" of the molecular origin 613 of the clonotypes with respect to the domestication of cats? 614 AUTHORS' RESPONSE -All strains are included (see l.136-138) for dating purposes and we did not 615 use the clone-censored dataset for this analysis. 616 3. Figure 3 needs to be radically overhauled . The ancestry plots suggest that Type I, II, III and Africa 4  617 do not exist as admixture clones, when in fact multiple studies (Boyle PNAS, 2006;Lorenzi, 618 NatComm, 2016;Zhang, 2017, Mol Biol Evol) clearly establish these lineages as admixtures. Hence, 619 the single color hue across each chromosome by clonotype belies truth. For example , Type II shares  620  chromosomes Ia and IV with Type I strains, and Type II shares chromosomes Ia, XI, and parts of Ib, III,  621 VI, VIIb, VIII, IX and XII with Type III. Figure 3 needs to be re-imagined, it fails to reflect truth, each 622 clonal strain is an admixture of different colors. 623 AUTHORS' RESPONSE -We understand the reviewer's concern. 624 In figure 3, we are defining the parental populations based on the results of global ancestry analyses 625 (mainly chromopainter). This analysis (Supplementary Figure 2) showed that most domestic New 626 World strains are sharing recent ancestry with the major intercontinental lineages on one hand and 627 the wild New World populations on the other hand. Therefore, Figure 3 succeeds in illustrating the 628 pattern of mixed ancestry of domestic New World strains which appear to be a mix between the 629 major intercontinental lineages and the wild New World populations. This is the key finding obtained 630 from this analysis. 631 We are aware that the major intercontinental lineages (types I, II, III and Africa 1) share common 632 ancestry for certain genomic loci and we are citing Boyle et al. in this sense (SI l.543). 633 When we aim to infer the parental populations of a hybrid population, we have to include in the 634 analysis a number of populations as putative parents of this hybrid population. However, in natural 635 populations of most species, having parental populations that are not related to each other by any 636 mean is very rare, so this is a "classical" concern with this kind of analyses. In the specific case of T. 637 gondii, the hybridizations that are involving the major intercontinental lineages are very recent and 638 too little time has elapsed for divergence to occur (we have produced NJ trees for each chromosome 639 and these trees show this pattern of absence of divergence). So it is likely that in certain genomic 640 positions, the software fails to distinguish between for example type II and type I or type II or type III. 641 However, this does not call into question the pattern of admixture that corresponds to the recent 642 history of encounter between to very divergent groups of populations (major intercontinental 643 lineages versus wild New World populations). 644 Another way to present the results could be to define only two ancestral groups: the major 645 intercontinental lineages one the one hand and the wild New World populations on the other hand 646 We could reset this analysis based on this division although we think this approach will hide many 647 interesting patterns. 648 The cut-offs also do not coincide with the analyses performed in Figure 4 and Supplemental Table 4.  649 For example, the HG12 strains WdUS01 and WdUS04 share the same color hue (green; Type II) on 650 chromosome III with two recombinant strains (WdUS02, WdUS03), but the analysis presented in 651 Supplemental Table 4 for these 4 isolates suggest that the two HG12 strains are at least 47K-177K 652 years distant from the two recombinant strains that share the same color at this chromosome. This 653 one example calls into question the entire dataset. 654 AUTHORS' RESPONSE -We used Ancestry_HMM for our local ancestry analyses. To our knowledge, 655 this is probably the only software that does not require genotypes from reference panels and that is 656 generalized to arbitrary ploidy, and is hence suitable for non-model haploid organisms such as T. 657 gondii. The software is based on a Hidden Markov Model and identifies at each position the most 658 probable ancestor for a hybrid strain among input ancestral populations and assign a probability to 659 this result. Who is the ancestor and who is the progeny depends on the assumptions obtained from 660 global ancestry analyses. The three (not two) HG12 strains to which the reviewer is referring were 661 assumed to be recombinant of a type II strain and another strain based on the result of global 662 ancestry analysis (mainly chromopainter). Ancestry_HMM found that type II is the most related 663 putative ancestral population to HG12 relatively to the other putative ancestral populations (this can 664 be expected as they belong to the same clade (Lorenzi et al., 2016) If the reviewer is referring to environmental strains, we will answer to this comment in two very 688 simple points: 689 1-All strains from this study (except four strains: DcTURKEY01 (Ankara LS1), DcUSA03(GT1), 690 DcUSA05(ME49), DcUSA04(M7741)) were sequenced less than 30 years after their isolation. 691 Isolated T. gondii strains are usually cryopreserved most of the time but the concern raised 692 by the reviewer is legitimate: we do not provide data about the number of passages 693 performed on these strains and new SNPs could have arisen during these passages. These 694 new SNPs could introduce some degree of bias in our calculations. Previously, we did not 695 know the mutation rate of T. gondii and therefore could not estimate to what extent strain 696 passages in the laboratory could introduce a bias in the TMRCA estimates. Fortunately, our 697 study provides for the first time an answer to this concern: we have estimated how many 698 SNPs can arise over a period of 30 years for a fast growing T. gondii strain subjected to an 699 intense regime of regular passages. We report in our study the results from an RH strain that 700 has been subjected to continuous in vivo culture during this long period. of Plasmodium in the study of T. gondii. We estimated the mutation rate of T. gondii to range 729 between 3.1x10 -9 to 11.7x10 -9 mutations per site per year, which is higher than the 1.7x10 -9 to 3.8x10 -730 9 estimated for Plasmodium. 731 5. The data estimates presented in Figure 4 suggest that the time between the two most divergent 732 genomes of Type II is significantly greater than for the other clonotypes, which they argue are more 733 recently derived. However, this result more likely reflects their bias in sampling. They had 48 Type II 734 strains to draw from, whereas they only had 3 Type I strains (Supplemental Table 8). Importantly, 735 there existed about 500 SNPs between the 3 Type I strains. Assuming the accumulation of SNPs is 736 similar among the different clonotypes (across time), 3 goes into 48 sixteen times (or 500x16=8000 737 SNPs) which is nearly equivalent to the number of SNPs that separate the two most divergent Type II 738 lineages. Which may suggest that Type I is as OLD as Type II and that the Figure 4 analysis is 739 potentially inaccurate and prone to an oversampling bias. 740 AUTHORS' RESPONSE -We totally agree with the reviewer on this point and we have already 741 clarified it in our manuscript l.265-268 (now l.440-442): "Note that inclusion of additional isolates 742 from the same or different geographic areas could alter these estimates by revealing more ancient 743 divergence times between strains of each respective lineage. This is particularly true for type I for 744 which only three samples were available." We have transferred these two sentences to the 745 discussion. 746 However, we would like to clarify a crucial point: 747 Lack of samples can only lead to an underestimation of the time of emergence of a given lineage (not 748 an overestimation). Therefore, our results provide strong support of an emergence of type II lineage 749 before domestication (12,980-48,988 years) and an emergence of type I, type III and Africa 1 before 750 the introduction of the domestic cat in the Americas. This is why we draw this conclusion l.437-440: 751 "Importantly, these domestic lineages emerged before dissemination of domestic cats to the New 752 World 500 years ago; it is hence likely that they emerged in the Old World." This is a crucial finding 753 for the consistency of our model which supports that Old World lineages "accompanied" domestic 754 cats in their expansion from the Old World to the New World. 755 One could always argue that having more samples will make the estimates more accurate but this a 756 classical concern that can only be corrected by isolating more and more samples. The most recent 757 example came from the study of our own species (Hublin et al., 2017 Nature): it was revealed in 2017 758 that we modern humans emerged at least (so maybe earlier we do not know) 100,000 years earlier 759 than previously believed. 760 6. The penetrance of the Chromosome Ia haplotype among successful strains of T. gondii that 761 expanded to represent a majority of infections in North America and Europe is certainly interesting 762 (and has been identified previously). While it is possible that the PBS analysis performed using the 763 strains in this study support a region on Chromosome !a as potentially relevant for the success of 764 these strains, the authors failed to prove that a gene in this region is responsible for the expansion of 765 these strains among domestic cats, as they strongly suggest. 766 AUTHORS' RESPONSE -In order to avoid lengthy repetitions, we invite the reviewer to refer to our 767 response to its second comment, in which we have already addressed precisely this concern. 768 Importantly, this analysis fails to include a large number of HG12 isolates, that are likewise highly 769 successful clones, that produce highly fecund infections in domestic cats, but do not share the same 770 chromosome Ia haplotype. 771 AUTHORS' RESPONSE -We hope our answer to the major comment number 1 of reviewer 3 provides 772 enough clarification regarding the specific case of HG12.
It is therefore incumbent on the authors to provide formal proof, that a gene in this region is both 774 necessary and sufficient to promote sexual reproduction in domestic cats to rationalize the success 775 of these clonotypes, as they report. 776 AUTHORS' RESPONSE -Evidence provided by this study is mainly genomic and 777 geographic/environmental. In addition, we provide for the first time a list of promising candidate 778 genes, enabling an important advance in the route of full evidence. We recall the fact that this is a 779 study of population genomics and not a mechanistic study, although it provides valuable data for 780 future purely mechanistic studies. 781 Minor Points: 782 1. Which is it? For Type II, Fig 2 lists 47 isolates, Supplemental Table 1 lists 50 isolates, Supplemental 783 Table 8 lists 48 isolates. Please be consistent. If strains are dropped from particular analyses, a 784 rational should be provided. 785 AUTHORS' RESPONSE -There is no mistake here and we justify the exclusion of these two strains. 786 The first one is DcFRANCE18 and was excluded due to poor sequencing depth (l.550) and the second 787 one (DcMARTINIQUE01) corresponds to a strain assumed to belong to type II lineage based on MS 788 markers, and that was found to be a recombinant strain with WGS (see the "Identifying clonal 789 lineages" section of SI). 790 2. Line 278 -"…TMRCA estimates indicate that introgressions of types I, II, II and Africa 1 into New 791 World populations have occurred…" I think the authors are suggesting here that Old World lineages 792 have recombined multiple times with New World strains to produce admixtures lines that exist as 793 chimeras -is that correct? 794 AUTHORS' RESPONSE -This is correct but the admixture pattern (Fig. 3) indicates that these 795 recombinations were not that frequent. 796 3. The authors renamed the vast majority of strains to conform with their "domestic" vs. "wild" 797 designation and geographic origin of the isolate. This was quite a distraction, to sort out which 798 designation was GT1, Me49, VEG, etc. 799 AUTHORS' RESPONSE -We understand the reviewer's concern and this was a difficult choice to 800 make. The original designations such as Me49 and VEG are well known for people in the field of 801 Toxoplasma. We renamed the strains to facilitate the understanding for non-specialists and to reach 802 out to a wider public of readers. Anyway the correspondence with old designations can be made in 803 Supplementary Table 1  804 The organization of the Supplemental Tables was difficult to infer. Is it possible to add the common 805 name to Supplemental Table 2, and also re-sort Supplemental corresponds to a strain assumed to belong to type III lineage based on MS markers, and that was 813 found to be a recombinant strain with WGS (see the "Identifying clonal lineages" section of SI). The 814 opposite was noticed for DcGabon02: It was a variant of type III on one marker based on MS marker, 815 but was found to be a true type III strain with WGS. 816 5. The authors propose a model in which rodents in the New World are multiply co-infected with 817 different strains of T. gondii to support the increased diversity detected in isolates from the New 818 World. Do the authors have evidence to support such a claim, or have studies been done that they 819 can reference that indicate such a high frequency of mixed infections to support their proposed 820 model? 821 AUTHORS' RESPONSE -The answer to this question can be found in our answer to a question from 822 reviewer number 2: A host infected with T. gondii develops good immunity (not 100% though) 823 against new infections. However, this immunity appears less efficient when the new infection 824 involves a highly divergent strain, allowing what is called superinfection to happen. The first study 825 that has described this phenomenon is a study by Elbez-Rubinstein et al., (2009). Here comes the 826 importance of defining what a divergent strain is and "divergence" is of course a relative concept. 827 According to our results and to previous results (Su et al., 2012;Lorenzi et al., 2016), it is clear for 828 example that wild Amazonian strains are highly divergent from the major domestic types (types I,II,III 829 and Africa 1). Our results also show that wild type 12 strains (RFLP lineage #5) from North America 830 are "moderately" divergent from type II strains. Domestic South American strains and wild South 831 American strains should not be considered one and the same, as the latter are a mixture of wild 832 South American strains and major domestic lineages. Domestic South American strains have 833 inherited large portions of their genome from types I, II, III and Africa 1 and this happened very 834 recently according to our dating estimates. Cross-immunity between these two groups is therefore 835 likely to occur, but this will likely be determined by the inheritance (or not) of alleles of genes 836 involved in immunological recognition. Anyway, it is important to recall that superinfected hosts 837 were rarely found in real life. This rarity fits well with our model, which supports rare recombinations 838 (see Fig.3